The present invention relates to a centrifuge microscope capable of observations in polarized light while applying a centrifuge force on the sample and observation methods performed using the centrifuge microscope.
Conventionally, centrifuge microscopes have been used for observing a sample while applying a centrifuge force by rotating a table on which the sample is placed at a high speed. An example of such centrifuge microscope is disclosed in the Jpn. Pat. Appln. KOKAI No. 63-250615. In the disclosed centrifuge microscope, a sample chamber in a rotor is arranged across the optical axis between a condenser lens and an objective lens. With the sample chamber rotating, the sample is illuminated with a pulsed light source. Since the transmitted light of the sample is accepted by the objective lens, a stationary observed image can be obtained regardless of the rotation of the sample chamber.
In the above mentioned disclosure, a stroboscope is used as the pulsed light source to illuminate the sample. The emission timing of the stroboscope is synchronized with the rotation of the rotor so that the emission takes place only when the sample chamber is on the optical axis between the condenser lens and the objective lens. In order to obtain a stationary image regardless of the rotation of the sample chamber, the emission time of the stroboscope needs to be sufficiently short. Otherwise, the observed image is blurred and provides low resolution.
Therefore, in the above-mentioned centrifuge microscope, in order to obtain stable stationary images even in the case where the emission time of the stroboscope cannot be sufficiently short, a parallel glass plate is provided between the objective lens and the rotor. The parallel plate is arranged orthogonally to the optical axis of the objective lens, and supported to be rotatable around an axis perpendicular to the tangent of the rotation of the sample chamber. The glass plate is tipped in synchronism with the rotation of the rotor in order to deviate the optical path such that the optical axis of light emitted from the sample in the sample chamber is always coincident with the optical axis of the objective lens. Accordingly, a bright image can be observed with good resolution without being blurred.
Conventionally, the following method is considered. For example, when the weakly birefringent mitotic spindle contained among highly birefringent yolk granules in an egg cell is observed as a sample in studies on cell division, the yolk granules with larger specific gravity are separated from the spindle with a centrifuge and the spindle is observed with a polarized light microscope after the cell is removed from the centrifuge. In this method, the percentage of centrifugally fragmented (yet viable) egg cells, containing spindles separated and gathered free from the yolk with the centrifuge, is less than 1%. Besides, since the specimen needs to be taken out from the centrifuge for observation, the yolk and the spindles co-mingle again, and thus it has been impossible to observe only the spindles with a polarized light microscope.
On the other hand, one can consider a method of enabling the imaging and measurement of the weak birefringence retardance, which reflects the concentration or the molecular orientation of microtubules comprising the spindles, with the specimen lying with in the centrifugal field, so as to separate yolk which has a larger specific gravity, using a centrifuge microscope. Observation of the specimen under centrifugal force would also provide a tool for studying the mechanism of cell division such as the measurement of the interaction between the chromosomes and the microtubules. However, observation of molecular orientation in the spindles, and visualization of the spindle fibers attached to the chromosmes, can be achieved only with a polarized light microscope capable of imaging weakly retarding objects, and the polarized light observation cannot be realized with a conventional centrifuge microscope.
In the case of conducting polarized light observation with the above-mentioned conventional centrifuge microscope, the parallel plate which is inserted between the objective lens and the rotor, for alleviating the conditions of emission time of the stroboscope, causes deterioration of polarizing characteristics in the polarized light observation, and thus effective polarized light observation cannot be conducted. Besides, in the case a xenon lamp or a flash lamp is used as the light source of the stroboscope, the quantity of light necessary for polarized light observation of weakly retarding objects cannot be provided and thus the polarized light observation cannot be achieved.
In addition, when an observation is conducted with the emission timing of the stroboscope synchronized with the rotation of the rotor, as the rotation speed of the rotor is changed the emission of the stroboscope and the rotation of the rotor can become out of synchronization so as to generate blurring in the observed image.
For the above-mentioned reasons, a centrifuge microscope capable of polarized light observation of weakly birefringent objects has so far not been available.